The fibroblast growth factor (FGF)-1 and FGF-2 proteins are likely to be important mediators of vascular cell growth in vivo since they (1) are potent angiogenic factors and smooth muscle cell mitogens and (2) are expressed by endothelial cells, smooth muscle cells and blood monocyte- derived macrophages. The possibility that FGFs may be involved in vascular disease has prompted numerous studies to identify strategies for effective inhibition of FGF biological activity. One such strategy is to interrupt the FGF mitogenic signaling pathway. Genes that are activated when FGF is added to quiescent cells may encode proteins that participate in this pathway; therefore, a mRNA differential display technique was used to obtain cDNA clones representing FGF-1-responsive genes expressed in murine NIH 3T3 fibroblasts. One of these genes, named Fnk for FGF-inducible kinase, is an immediate-early gene highly inducible by FGF-1, FGF-2, PDGF- BB, serum and phorbol ester. This gene is also transiently expressed in mouse liver after partial hepatectomy and in rat carotid artery after balloon injury. Nucleotide sequence analysis indicated that the Fnk cDNA encoded a novel serine/threonine protein kinase, with the catalytic domain occupying the amino-terminal half of the molecule. The predicted Fnk protein has approximately 49% amino acid sequence identity to the mouse Snk protein, which is also a putative serine/threonine kinase encoded by an mediate-early gene. Furthermore, Fnk is structurally-related to mammalian Plk and Drosophila polo, protein kinases required for cell cycle progression. The major objective of the research described in this application is to determine whether Fnk is a serine/threonine kinase involved in the FGF intracellular signaling pathway. Accordingly, the specific aims of this proposal are: 1) to determine whether Fnk protein, like Fnk mRNA, is transiently expressed in FGF-1-stimulated cells and to identify its subcellular location; 2) to determine whether Fnk is in fact a serine/threonine-specific protein kinase; 3) to determine whether Fnk is phosphorylated in FGF-1-stimulated cells, and if so, to identify the phosphorylation site(s) and determine whether phosphorylation alters enzymatic activity; and 4) to determine whether Fnk function is required for FGF-1-stimulated cell proliferation and whether Fnk itself is mitogenic. It is anticipated that these studies will provide novel information on the mechanism of FGF-1 and FGF-2 mitogenic signal transduction and may identify Fnk as a therapeutic target for antiproliferative therapy.